O₂-evolving chlorite dismutase as a tool for studying O ₂-utilizing enzymes

The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas's modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, in situ generation of O₂ at concentrations far exceeding 2 mM. This method was used to define the O₂ concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis, in which the enzyme's Mn^IV/Fe^III cofactor forms from a Mn^II/Fe^II complex and O₂ via a Mn ^IV/Fe^IV intermediate, at effective O₂ concentrations as high as ∼10 mM. With a more soluble receptor, myoglobin, an O₂ adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C-H-bond-cleaving Fe^IV-oxo complex, J, in taurine:α-ketoglutarate dioxygenase and superoxo-Fe₂ ^III/III complex, G, in myo-inositol oxygenase, and the tyrosyl-radical-generating Fe₂^III/IV intermediate, X, in Escherichia coli RNR, were all accumulated to yields more than twice those previously attained. This means of in situ O₂ evolution permits a >5 mM "pulse" of O₂ to be generated in <1 ms at the easily accessible Cld concentration of 50 μM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O₂ addition steps.